Farnesyltransferase inhibitor lonafarnib suppresses respiratory syncytial virus infection by blocking conformational change of fusion glycoprotein

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.

1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: ELECTRON MICROSCOPY The reported resolution of this entry is 3.17 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic.The table shows the number of entries on which the scores are based.The table below summarises the geometric issues observed across the polymeric chains and their fit to the map.The red, orange, yellow and green segments of the bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively.A grey segment represents the fraction of residues that are not modelled.The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5%

Metric
The upper red bar (where present) indicates the fraction of residues that have poor fit to the EM map (all-atom inclusion < 40%).The numeric value is given above the bar. 2 Entry composition i ○

Mol Chain Length Quality of chain
There are 4 unique types of molecules in this entry.The entry contains 12307 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called Fusion glycoprotein F0,Fibritin.3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry.The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and atom inclusion in map density.Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more.A red diamond above a residue indicates a poor fit to the EM map for this residue (all-atom inclusion < 40%).Stretches of 2 or more consecutive residues without any outlier are shown as a green connector.Residues present in the sample, but not in the model, are shown in grey.

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Page 10 Full wwPDB EM Validation Report (*For Manuscript Review*) EMD-37210, 8KG5 • Molecule 1: Fusion glycoprotein F0,Fibritin Chain C: • Molecule 2: D25 heavy chain Chain D: There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms).The all-atom clashscore for this structure is 12.
All (296) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude.There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all PDB entries followed by that with respect to all EM entries.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.

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Page 22 Full wwPDB EM Validation Report (*For Manuscript Review*) EMD-37210, 8KG5

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all PDB entries followed by that with respect to all EM entries.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.

RNA i ○
There are no RNA molecules in this entry.

Non-standard residues in protein, DNA, RNA chains i ○
There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligand geometry i ○
3 ligands are modelled in this entry.
In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the Chemical Component Dictionary.The Link column lists molecule types, if any, to which the group is linked.The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).In the following table, the Chirals column lists the number of chiral outliers, the number of chiral centers analysed, the number of these observed in the model and the number defined in the Chemical Component Dictionary.Similar counts are reported in the Torsion and Rings columns.'-' means no outliers of that kind were identified.

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Page The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest.In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included.For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier.
Any bond that is central to one or more torsion angles identified as an outlier by Mogul will be highlighted in the graph.For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identified by Mogul is calculated over all ring torsion angles.If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identified rings is also greater than 60 degrees, then that ring is considered an outlier.
The outliers are highlighted in purple.The color gray indicates Mogul did not find sufficient equivalents in the CSD to analyse the geometry.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry.6 Map visualisation i ○ This section contains visualisations of the EMDB entry EMD-37210.These allow visual inspection of the internal detail of the map and identification of artifacts.
Images derived from a raw map, generated by summing the deposited half-maps, are presented below the corresponding image components of the primary map to allow further visual inspection and comparison with those of the primary map.

Orthogonal projections
The images above show the map projected in three orthogonal directions.The images above show central slices of the map in three orthogonal directions.The images above show the 3D surface view of the map at the recommended contour level 0.1.These images, in conjunction with the slice images, may facilitate assessment of whether an appropriate contour level has been provided.

X Y Z
These images show the 3D surface of the raw map.The raw map's contour level was selected so that its surface encloses the same volume as the primary map does at its recommended contour level.

Mask visualisation i ○
This section was not generated.No masks/segmentation were deposited.7 Map analysis i ○ This section contains the results of statistical analysis of the map.

Map-value distribution i ○
The map-value distribution is plotted in 128 intervals along the x-axis.The y-axis is logarithmic.A spike in this graph at zero usually indicates that the volume has been masked.The images above show the model with each residue coloured according its Q-score.This shows their resolvability in the map with higher Q-score values reflecting better resolvability.Please note: Q-score is calculating the resolvability of atoms, and thus high values are only expected at resolutions at which atoms can be resolved.Low Q-score values may therefore be expected for many entries.9.5 Map-model fit summary i ○ The table lists the average atom inclusion at the recommended contour level (0.1) and Q-score for the entire model and for each chain.
The images above show the largest variance slices of the map in three orthogonal directions.The images above show the map standard deviation projections with false color in three orthogonal directions.Minimum values are shown in green, max in blue, and dark to light orange shades represent small to large values respectively.
estimate i ○The volume at the recommended contour level is 74 nm 3 ; this corresponds to an approximate mass of 67 kDa.The volume estimate graph shows how the enclosed volume varies with the contour level.The recommended contour level is shown as a vertical line and the intersection between the line and the curve gives the volume of the enclosed surface at the given level.averaged power spectrum i ○ *Reported resolution corresponds to spatial frequency of 0.315 Å −1 Correlation (FSC) is the most commonly used method to estimate the resolution of single-particle and subtomogram-averaged maps.The shape of the curve depends on the imposed symmetry, mask and whether or not the two 3D reconstructions used were processed from a common reference.The reported resolution is shown as a black line.A curve is displayed for the half-bit criterion in addition to lines showing the 0.143 gold standard cut-off and 0.5 cut-off.8.1 FSC i ○ *Reported resolution corresponds to spatial frequency of 0.315 Å −1

9
Map-model fit i ○This section contains information regarding the fit between EMDB map EMD-37210 and PDB model 8KG5.Per-residue inclusion information can be found in section 3 on page 9.9.1 Map-model overlayi ○ X Y ZThe images above show the 3D surface view of the map at the recommended contour level 0.1 at 50% transparency in yellow overlaid with a ribbon representation of the model coloured in blue.These images allow for the visual assessment of the quality of fit between the atomic model and the map.

9. 3
Atom inclusion mapped to coordinate modeli ○ X Y ZThe images above show the model with each residue coloured according to its atom inclusion.This shows to what extent they are inside the map at the recommended contour level (0.1). inclusion i ○At the recommended contour level, 84% of all backbone atoms, 78% of all non-hydrogen atoms, are inside the map.

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Bond lengths and bond angles in the following residue types are not validated in this section: 336The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).
table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively.The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity.The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes.
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8.2 Resolution estimates i○ Resolution estimate based on FSC curve calculated by comparison of deposited half-maps.The value from deposited half-maps intersecting FSC 0.143 CUT-OFF 4.03 differs from the reported value 3.17 by more than 10 % *